MANUALTSA® Research Reagents
Caution: For Laboratory Use. A research chemical for research purposes only.

TSA Plus Fluorescence Kits

Product Information
Storage:

Store kits in the dark at 4ºC.

Stability:

The components in the kits are stable for a minimum of 3 months under proper storage
conditions.

Application:

TSA Plus Fluorescence kits are intended for high sensitivity detection in immunohistochemistry(IHC), immunocytochemistry (ICC) and in situ hybridization (ISH) experiments. Final detection
may be fluorescent or chromogenic (with TSA Plus Fluorescein kits only).

FOR RESEARCH USE ONLY.

Safety Note

All reagents are classified as nonhazardous. However, it is strongly recommended to wear
disposable gloves and safety glasses while working. Thorough washing of hands after handling is
also recommended.

Quality Control

We certify that QC results of these reagents meet our quality release criteria.

What is TSA technology?
®

TSA from PerkinElmer is a technology that improves fluorescent and chromogenic signals by up to 100-fold whileallowing reduced consumption of primary antibodies or probes. TSA Plus is an optimized and enhanced version that is up
to 20 times more sensitive than standard tyramide reagents.
TSA Plus Fluorescence Kits use horseradish peroxidase (HRP) to catalyze covalent deposition of fluorophores directly
adjacent to the immobilized enzyme. The labeling reaction is quick (less than 10 minutes) and deposited labels can thenbe viewed directly using standard or confocal microscopy. TSA Plus Fluorescein may also be used in combination with
anti-fluorescein enzyme conjugates and appropriate chromogenic substrates for brightfield microscopy.
The use of TSA reagents results in a significant increase in sensitivity over standard detection methods, while maintaining
specificity and resolution. Moreover, TSA reagents allow drastically reduced consumption of primary antibody or probe.
TSA Plus Fluorescence kits are ideal for multiplexed IHC or ISH in combination. The Opal™ protocol allows simultaneous
measurement of 6 or more biomarkers in FFPE tissue. For more information, please see www.perkinelmer.com/opal.

1Material Provided
Format*Catalog #50-150 slides

NEL741001KT 

1X Plus Amplification Diluent (15 mL)
Fluorescein Plus Amplification Reagent (2 tubes, add 150 µL DMSO / tube)

250-750 slides

NEL741B001KT 

1X Plus Amplification Diluent (5 x 15 mL)
Fluorescein Plus Amplification Reagent (10 tubes, add 150 µL DMSO / tube)

25-75 slides

NEL741E001KT 

1X Plus Amplification Diluent (7.5 mL)
Fluorescein Plus Amplification Reagent (1 tube, add 150 µL DMSO)

50-150 slides

NEL742001KT 

1X Plus Amplification Diluent (15 mL)
TMR Plus Amplification Reagent (2 tubes, add 150 µL DMSO / tube)

250-750 slides

NEL742B001KT 

1X Plus Amplification Diluent (5 x 15 mL)
TMR Plus Amplification Reagent (10 tubes, add 150 µL DMSO / tube)

50-150 slides

NEL744001KT 

1X Plus Amplification Diluent (15 mL)
Cyanine 3 Plus Amplification Reagent (2 tubes, add 150 µL DMSO / tube)

250-750 slides

NEL744B001KT 

1X Plus Amplification Diluent (5 x 15 mL)
Cyanine 3 Plus Amplification Reagent (10 tubes, add 150 µL DMSO / tube)

25-75 slides

NEL744E001KT 

1X Plus Amplification Diluent (7.5 mL)
Cyanine 3 Plus Amplification Reagent (1 tube, add 150 µL DMSO)

50-150 slides

NEL763001KT 

1X Plus Amplification Diluent (15 mL)
Cyanine 3.5 Plus Amplification Reagent (2 tubes, add 150 µL DMSO / tube)

250-750 slides

NEL763B001KT 

1X Plus Amplification Diluent (5 x 15 mL)
Cyanine 3.5 Plus Amplification Reagent (10 tubes, add 150 µL DMSO / tube)

25-75 slides

NEL763E001KT 

1X Plus Amplification Diluent (7.5 mL)
Cyanine 3.5 Plus Amplification Reagent (1 tube, add 150 µL DMSO)

50-150 slides

NEL745001KT 

1X Plus Amplification Diluent (15 mL)
Cyanine 5 Plus Amplification Reagent (2 tubes, add 150 µL DMSO / tube)

250-750 slides

NEL745B001KT 

1X Plus Amplification Diluent (5 x 15 mL)
Cyanine 5 Plus Amplification Reagent (10 tubes, add 150 µL DMSO / tube)

25-75 slides

NEL745E001KT 

1X Plus Amplification Diluent (7.5 mL)
Cyanine 5 Plus Amplification Reagent (1 tube, add 150 µL DMSO)

50-150 slides

NEL766001KT 

1X Plus Amplification Diluent (15 mL)
Cyanine 5.5 Plus Amp Reagent (2 tubes, add 150 µL DMSO / tube)

250-750 slides

NEL766B001KT 

1X Plus Amplification Diluent (5 x 15 mL)
Cyanine 5.5 Plus Amplification Reagent (10 tubes, add 150 µL DMSO / tube)

25-75 slides

NEL766E001KT 

1X Plus Amplification Diluent (7.5 mL)
Cyanine 5.5 Plus Amplification Reagent (1 tube, add 150 µL DMSO)TSA Plus Cyanine 3 /Cyanine 5

50-150 slides

NEL752001KT 

TSA Plus Cyanine 3 /Fluorescein50-150 slides

NEL753001KT

TSA Plus Cyanine 5 /Fluorescein50-150 slides

NEL754001KT

TSA Plus Fluorescein /Tetramethylrhodamine

50-150 slides

NEL756001KT

TSA Plus Fluorescence
Palette Kit

10-35 slides

NEL760001KT

1X Plus Amplification Diluent (2 x15 mL)
Cyanine 3 Plus Amplification Reagent (2 tubes, add 150 µL DMSO / tube)
Cyanine 5 Plus Amplification Reagent (2 tubes, add 150 µL DMSO / tube)
1X Plus Amplification Diluent (2 x15 mL)
Cyanine 3 Plus Amplification Reagent (2 tubes, add 150 µL DMSO / tube)
Fluorescein Plus Amplification Reagent (2 tubes, add 150 µL DMSO / tube)
1X Plus Amplification Diluent (2 x15 mL)
Cyanine 5 Plus Amplification Reagent (2 tubes, add 150 µL DMSO / tube)
Fluorescein Plus Amplification Reagent (2 tubes, add 150 µL DMSO / tube)
1X Plus Amplification Diluent (2 x15 mL)
TMR Plus Amplification Reagent (2 tubes, add 150 µL DMSO / tube)
Fluorescein Plus Amplification Reagent (2 tubes, add 150 µL DMSO / tube)
1X Plus Amplification Diluent (2 x15 mL)
Fluorescein Plus Amplification Reagent (1 tubes, add 60 µL DMSO)TMR Plus Amplification Reagent (1 tube, add 60 µL DMSO)
Cyanine 3 Plus Amplification Reagent (1 tube, add 60 µL DMSO)
Cyanine 5 Plus Amplification Reagent (1 tube, add 60 µL DMSO)TSA Plus Fluorescein

TSA Plus
Tetramethylrhodamine

TSA Plus Cyanine 3

TSA Plus Cyanine 3.5

TSA Plus Cyanine 5

TSA Plus Cyanine 5.5

Kit Components

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*The format of the kit is based on 100 - 300 µL per slide of TSA Plus Working Solution (see page 3).

2

Reagents and Materials
Critical Reagents Required but not Supplied HRP-labelled reagent to drive the signal amplification reaction is required. For example:o Anti-digoxigenin-HRP for use with digoxigenin labeled probes or antibodies (cat. no. NEF832001EA)o Anti-fluorescein-HRP for use with fluorescein labeled probes or antibodies (cat. no. NEF710001EA)o HRP-conjugated anti-species secondary antibody
 Anti-rabbit IgG (goat) HRP (cat. no. NEF812001EA)
 Anti-mouse IgG (goat) HRP (cat. no. NEF822001EA) DMSO (molecular biology or HPLC grade) Buffer components and detergents such as PBS, Triton-X100, Tween-20
 Blocking Reagent (cat. no. FP1012 or equivalent)

Solutions to prepare
The following buffers and reagents are required for slide preparation and signal amplification
TSA Plus Stock Solution
TSA Plus Amplification Reagent is supplied as a solid. Each vial must be reconstituted with molecular biology or HPLC grade
DMSO to make TSA Plus Stock Solution. TSA Plus Stock Solution is stable for at least 3 months when stored at 4ºC. (Note:DMSO freezes at 4ºC; therefore, thaw Stock Solution before each use). Here are recommended dissolution volumes forstock solutions.
 For the TSA Plus Fluorescence Palette Kit (NEL760001KT), add 60 µL DMSO to each tube of Amplification
Reagent.
 Add 150 µL DMSO per tube Amplification Reagent for all other TSA Plus Fluorescence Kits.
TNT Wash Buffer
0.1 M TRIS-HCl, pH 7.5
0.15 M NaCl
0.05% Tween®20
Other wash buffers (such as PBS) may be used. Substitution of 0.3% Triton X-100 for the 0.05% Tween-20 is also possible.
Users should validate the use of alternative wash buffers with their own systems.
TNB Blocking Buffer
0.1 M TRIS-HCl, pH 7.5
0.15 M NaCl
0.5% Blocking Reagent (available separately, catalog number FP1012)
Add Blocking Reagent slowly in small increments to buffer while stirring. Heat gradually to 55°C with continuous stirring tocompletely dissolve the Blocking Reagent. (This should take no longer than 30-60 minutes.) The solution will appear milky.
Bring to room temperature before using. Aliquot and store at -20°C for long term use.
TSA Plus Working Solution
Before each procedure, dilute TSA Plus Stock Solution1:50 in 1X Amplification Diluent to make TSA Plus Working Solution.
Approximately 100-300 µL of TSA Plus Working Solution is required per slide. Discard any unused portion of TSA Plus
Working Solution.

3

Recommendations


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
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


Do not let slides dry out between steps.
A humidified chamber is recommended for all incubation steps (i.e., a damp paper towel in a covered box).
Drain off as much of the incubation solutions as possible, before the addition of the next solution, to prevent reagent
dilution and uneven staining. Blot area around, but not on, tissue section using a tissue.
Be sure to use enough volume of each reagent to completely cover sections or cells.
Optional: Use of coverslips may reduce reagent evaporation, especially during steps which require long incubation atelevated temperatures (such as probe hybridization). However care must be taken upon removal to prevent damage
to tissues or cells.
If your assay includes streptavidin conjugates, check for endogenous biotin which may be a source of non-specificbackground.
If there is too much signal, dilute the primary antibody, probe or HRP conjugate further. TSA Plus kits are designed
for use at 1:50 dilution of TSA Plus Stock Solution for optimal results.
First time users should apply TSA to a proven system.

Quenching Endogenous Peroxidase
Activation and covalent binding of TSA reagent is catalyzed by peroxidase. Endogenous peroxidase will be a source of
background, and should be quenched if present. Users should establish the need for doing this and optimal methodology
specific to the tissues or cells being stained. Options include: 0.3% H2O2 to 3% H2O2 in PBS, incubation for 10 to 60 minutes
 0.3% H2O2 to 3% H2O2 in Methanol, incubation for 10 to 60 minutes
For paraffin-embedded tissues, quenching can be done after dewaxing and alcohol rehydration but before the protease
digestion step. For frozen tissue or cell preps, quenching can be done following fixation and before the protease
digestion step. After quenching wash with TNT or 1X PBS buffer for 5 minutes. See Li et al from references section for
more suggestions.

TSA-ISH Optimization
Inadequate optimization of probe and HRP conjugate dilution may be a source of high background and reduced signal.
TSA is ideal for detection of targets that have weak signals using standard detection methods. If the signal is strong withstandard detection methods, dilute the probe until the signal begins to disappear and then proceed with TSA
optimization.
Probe Optimization
 Slide 1: use same probe dilution as standard method.
 Slide 2: 5-fold dilution from slide 1
 Slide 3: 2-fold dilution from slide 2 (further dilution may be necessary) Slide 4: negative control (probe omitted)
HRP Conjugate Dilution
 SA-HRP included with TSA Systems: 1:100 dilution
 Streptavidin-HRP (catalog number NEL750001EA): 1:250-1:1000 dilution
 Anti-fluorescein-HRP (catalog number NEF710001EA): 1:100-1:500 dilution
 Anti-DNP-HRP (catalog number FP1129): 1:100-1:500 dilution
 Anti-digoxigenin-HRP (catalog number NEF832001EA): 1:500-1:5000 dilution
 HRP reagents from other manufacturers: Begin with recommended range for slide applications. Further dilution
may be needed4

TSA-ISH Protocol Overview
Standard
nonradiometric
ISH
Blocking Step
Incorporationof HRP
TSA
Amplification

Visualization

Quench endogenous peroxidase activity (if needed)
Tissue permeabilization (if needed)
Probe hybridization
Post-hybridization stringency washes↓
Block slides for 30 min. in TNB buffer at room temperature↓
Incubate slides with appropriate HRP reagent (anti-DIG-HRP, SA-HRP, etc.) for 30 min. at room
temperature↓
Wash slides 3X for 5 min. in TNT buffer at room temperature Incubate in TSA Plus working solution
for 3 to 10 min. at room temperature↓
Wash slides 3X for 5 min. in TNT buffer at room temperature↓↓
Fluorescent
Chromogenic (TSA Plus Fluorescein Only)
Counterstain and mount for fluorescence
Microscopy

Incubate slides in anti-fluorescein-HRP or
anti-fluorescein-AP for 30 min. at room
temperature↓
Wash slides 3X for 5 min. in TNT buffer atroom temperature↓
Add appropriate chromogen, counterstain andmount for microscopy

Step by Step TSA-ISH Protocol
Step 1: Slide Preparation
Prepare tissues or cells using standard fixation and embedding techniques. Dewax and rehydrate slides according tostandard procedures.
Step 2: Standard Non-radioactive ISH
Follow standard non-radioactive in situ hybridization techniques. Include tissue permeabilization (if needed) andquenching of endogenous peroxidase activity (if needed). Probe hybridization (with digoxigenin, biotin, DNP, or
fluorescein-labeled probes) should be done using at concentration determined in optimization studies followed by posthybridization stringency washes.
NOTE: Always run an unamplified control slide and an amplified negative control slide with each experiment.
Step 3: Blocking Step
Cover tissue sections with TNB buffer (or other validated blocking buffer) and incubate slides in a humidified chamber for
30-60 minutes at room temperature or at 4ºC overnight.

5Step 4: Introduction of HRP
Incubate slides for 30 minutes at room temperature or at 4ºC overnight in a humidified chamber with appropriate HRPlabeled reagent. Use adequate reagent volume to cover the tissue section, generally 100-300 µL per slide.
 For DIG-labeled probes, use anti-digoxigenin-HRP (catalog number NEF832001EA) diluted in TNB Buffer (orother validated blocking buffer) to concentration determined in optimization studies.
 For biotin-labeled probes, use SA-HRP (catalog number NEL750001EA) diluted in TNB Buffer (or other validated
blocking buffer) to concentration determined in optimization studies.
 For fluorescein-labeled probes, use anti-fluorescein-HRP (catalog number NEF710001EA) diluted in TNB Buffer(or other validated blocking buffer) to concentration determined in optimization studies.
 For DNP-labeled probes, use anti-DNP-HRP (catalog number FP1129) diluted in TNB Buffer (or other validated
blocking buffer) to concentration determined in optimization studies.
Step 5: Wash
Wash the slides 3X for 5 minutes each in TNT Buffer (or other validated wash buffer) at room temperature with agitation.
Step 6: Amplification
Pipette TSA Plus Working Solution onto each slide. Use enough working solution to completely cover the tissue section,
generally 100-300 µL per slide. Incubate the slides at room temperature for 3 to 10 minutes.
Step 7: Wash
Wash the slides 3X for 5 minutes each in TNT buffer (or other validated wash buffer) at room temperature with agitation.
Step 8: Visualization of Deposited Fluorophores
For fluorescent detection, counterstain and mount for fluorescence microscopy. (See page 10 table, Fluorophore
Excitation and Emission Maxima.)
For chromogenic detection with TSA Plus Fluorescein, incubate slides with anti-fluorescein-HRP (NEF710001EA) or antifluorescein-AP (NEF709001PK) followed by detection with an appropriate chromogenic substrate.

TSA-IHC Optimization
Inadequate optimization of primary antibody and HRP conjugate dilution may be a source of high background andreduced signal.
TSA is ideal for detection of targets that have weak signals with standard detection methods. It enables use of much
more dilute antibody solutions for improved specificity. If the signal is strong with standard detection methods, dilute theprimary antibody until the signal begins to disappear and then proceed with TSA optimization.
Primary Antibody Optimization
 Slide 1: use same antibody dilution as standard method.
 Slide 2: 5-fold dilution from slide 1
 Slide 3: 5-fold dilution from slide 2
 Slide 4: 5-fold dilution from slide 3 (further dilution may be necessary) Slide 4: negative control (primary antibody omitted)
HRP Conjugate Dilution
 SA-HRP included with TSA Systems: 1:100 dilution
 Anti-mouse-HRP (catalog number NEF822001EA): 1:500 to 1:2000 dilution
 Anti-rabbit-HRP (catalog number NEF812001EA): 1:500 to 1:2000 dilution
 Streptavidin-HRP (catalog number NEL750001EA): 1:1250-1:2500 dilution
 Anti-fluorescein-HRP (catalog number NEF710001EA): 1:100-1:500 dilution
 HRP reagents from other manufacturers: Begin with recommended range for slide applications. Further dilution
may be needed
6

TSA-IHC Protocol Overview
Quench endogenous peroxidase activity (if needed)↓
Block slides for 30 min. in TNB buffer at room temperatureStandard
IHC

Incorporationof HRP

TSA
Amplification

Visualization↓
Incubate slides in primary antibody for 30-60 minutes at room temperature↓
Wash slides 3X for 5 min. in TNT buffer at room temperature↓
Incubate slides in biotinylated secondary
antibody 30-60 min. at room temperature
Incubate slides in HRP labeled secondary
Wash slides 3X for 5 min. in TNT buffer atorantibody for 30 min. at room temperatureroom temperature
Incubate slides in SA-HRP for 30 min. at room
temperature↓
Wash slides 3X for 5 min. in TNT buffer at room temperature↓
Incubate in TSA Plus Working Solution for 3 to 10 min. at room temperature↓
Wash slides 3X for 5 min. in TNT buffer at room temperature↓
Fluorescent
Chromogenic (TSA Plus Fluorescein Only)
Counterstain and mount for fluorescencemicroscopy

Incubate slides in anti-fluorescein-HRP or
anti-fluorescein-AP for 30 min. at room
temperature↓
Wash slides 3X for 5 min. in TNT buffer atroom temperature↓
Add appropriate chromogen, counterstain andmount for microscopy

Step by Step TSA-IHC Protocol
Step 1: Slide Preparation
Prepare tissues or cells for detection with TSA using standard fixation and embedding techniques. Dewax and rehydrateusing standard protocols. Quench endogenous peroxidase activity if necessary.
NOTE: Always run an unamplified control slide and an amplified negative control slide with each experiment.
Step 2: Blocking Step
Cover tissue sections with TNB buffer (or other validated blocking buffer) and incubate slides in a humidified chamber for
30-60 minutes at room temperature or at 4ºC overnight.
Step 3: Primary Antibody Incubation
Drain off the blocking buffer and apply primary antibody, diluted in TNB Buffer (or other validated blocking buffer).
Incubate the primary antibody preparation per the manufacturer's instructions regarding incubation time and temperature
requirements. Use enough volume to completely cover the tissue section (generally 100-300 µL per slide) at theconcentration determined in optimization studies.
7

Step 4: Wash
Wash the slides 3X for 5 minutes each in TNT Buffer (or other validated wash buffer) at room temperature with agitation.
Step 5: Introduction of HRP
Incubate slides for 30 minutes at room temperature or at 4ºC overnight in a humidified chamber with appropriate HRPlabeled reagent. Use adequate reagent volume to cover the tissue section, generally 100-300 µL per slide. Options
include.
 HRP labeled secondary antibody diluted in TNB Buffer (or other validated blocking buffer).
 100-300 µL of biotinylated secondary antibody diluted in TNB Buffer (or other validated blocking buffer). Incubate
30-60 minutes in a humidified chamber. Wash the slides for 3 X 5 minutes TNT buffer at room temperature withagitation. Follow by 100-300 µL of SA-HRP diluted in TNB Buffer. Use SA-HRP at 1:100 dilution, or at 1:2000 if
using PerkinElmer Cat. # NEL750001EA.
 When using alternative suppliers, reagents should be optimized for use with TSA starting with manufacturer's
recommended dilutions. Incubate slides in a humidified chamber for 30-60 minutes at room temperature or at
4ºC overnight.
Step 6: Wash
Wash the slides 3X for 5 minutes each in TNT Buffer (or other validated wash buffer) at room temperature with agitation.
Step 7: Amplification
Pipette 100-300 µL of TSA Plus Working Solution onto each slide. Incubate the slides at room temperature for 3 to 10
minutes.
Step 8: Wash
Wash the slides 3X for 5 minutes each in TNT Buffer (or other validated wash buffer) at room temperature with agitation.
Step 9: Visualization of Deposited Fluorophores
For fluorescent detection, counterstain and mount for fluorescence microscopy. (See page 10 table, Fluorophore
Excitation and Emission Maxima.)
For chromogenic detection with TSA Plus Fluorescein, incubate slides with anti-fluorescein-HRP (NEF710001EA) or antifluorescein-AP (NEF709001PK) followed by detection with an appropriate chromogenic substrate.

8Troubleshooting
Technical Support Resources Assay Support Knowledge Base: Email: Telephoneo USA toll-freeo EU toll-freeo Finland toll-freeo China toll-free

www.perkinelmer.com/askTSA
global.techsupport@perkinelmer.com
800-762-4000
00800 33 29 0000
999 800 33 29 0000
800 820 5046

ISH Troubleshooting
PROBLEM

REMEDY

Low Signal

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

Optimize probe concentration.
Titer HRP conjugate to determine optimum concentration for signal amplification.
Add tissue permeabilization step to facilitate penetration of reagents.
Lengthen incubation time for TSA Plus Working Solution.

Excess Signal

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

High
Background

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Decrease concentration of HRP conjugate introduced prior to amplification.
Decrease probe concentration.
Decrease TSA Plus Working Solution incubation time.
Decrease concentration of anti-fluorescein-enzyme conjugate used for chromogenic
visualization.
Decrease probe concentration.
Decrease concentration of HRP conjugate.
Check for endogenous biotin (if using streptavidin conjugates)
Shorten chromogenic development time.
Lengthen endogenous peroxidase quenching step.
Filter buffers.
Increase number and/or length of washes.
Nonqualified or contaminated blocking reagent used. Use PerkinElmer Blocking Reagent(FP1020 or FP1012).

IHC Troubleshooting
PROBLEM

REMEDY

Low Signal



Excess Signal





High
Background









Titer primary and/or secondary antibodies to determine optimum concentration for signal
amplification
Lengthen incubation time for TSA Plus Working Solution.
Use antigen retrieval techniques to unmask the target.
Decrease concentration of primary and/or secondary antibody or HRP conjugates.
Decrease TSA Plus Working Solution incubation time.
Decrease concentration of anti-fluorescein-enzyme conjugate used for chromogenic
visualization.
Filter buffers
Decrease concentration of primary and/or secondary antibody or HRP conjugates.
Lengthen endogenous peroxidase quenching step.
Check for endogenous biotin (if using streptavidin conjugates)
Increase number and/or length of washes.
Shorten chromogenic development time.
Nonqualified or contaminated blocking reagent used. Use PerkinElmer Blocking Reagent(FP1020 or FP1012).

9

Selected References
Brend, Tim, and Scott A. Holley. "Zebrafish whole mount high-resolution double fluorescent in situ hybridization." Journalof visualized experiments: JoVE 25 (2009).
Brown, Jason, Hallie Wimberly, Donald R. Lannin, Christian Nixon, David L. Rimm, and Veerle Bossuyt. "Multiplexed
Quantitative Analysis of CD3, CD8, and CD20 Predicts Response to Neoadjuvant Chemotherapy in Breast
Cancer."Clinical Cancer Research (2014): clincanres-1622.
Lee, Seung-won, Song Eun Lee, Seong Hyuk Ko, Eun Kyoung Hong, Kwang Il Nam, Kei-ichiro Nakamura, Shuhei
Imayama et al. "Introduction of tyramide signal amplification (TSA) to pre-embedding nanogold-silver staining at theelectron microscopic level." Journal of Histochemistry & Cytochemistry 53, no. 2 (2005): 249-252.
Lein, Ed S., Michael J. Hawrylycz, Nancy Ao, Mikael Ayres, Amy Bensinger, Amy Bernard, Andrew F. Boe et al.
"Genome-wide atlas of gene expression in the adult mouse brain." Nature 445, no. 7124 (2006): 168-176.
Liu, Gang, Sejal Amin, Nataly N. Okuhama, Guoning Liao, and Lisa A. Mingle. "A quantitative evaluation of peroxidase
inhibitors for tyramide signal amplification mediated cytochemistry and histochemistry." Histochemistry and cellbiology 126, no. 2 (2006): 283-291.
Silahtaroglu, Asli N., Dorrit Nolting, Lars Dyrskjøt, Eugene Berezikov, Morten Møller, Niels Tommerup, and Sakari
Kauppinen. "Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic
acid probes and tyramide signal amplification." Nature protocols 2, no. 10 (2007): 2520-2528.
Stack, Edward C., Chichung Wang, Kristin Roman, and Clifford C. Hoyt. "Multiplexed immunohistochemistry, imaging,and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex
analysis." Methods (2014).
Zaidi, Aliya U., Hideki Enomoto, Jeffrey Milbrandt, and Kevin A. Roth. "Dual fluorescent in situ hybridization andimmunohistochemical detection with tyramide signal amplification." Journal of Histochemistry & Cytochemistry 48, no. 10(2000): 1369-1375.

TSA Fluorophore Excitation and Emission Maxima
Fluorophore

Excitation

Emission

Coumarin

402 nm443 nmFluorescein

494 nm

517 nmTetramethylrhodamine

550 nm

570 nmCyanine 3

550 nm

570 nmCyanine 3.5

581 nm

596 nmCyanine 5

648 nm

667 nmCyanine 5.5

673 nm692 nm10

Complementary Products
3g

FP1020

10 g

FP1012Anti-rabbit IgG (goat) HRP

1 mL

NEF812001EA

Anti-rabbit IgG (goat) HRP

100 µLNEF812E001EA

Anti-mouse IgG (goat) HRP

1 mL

NEF822001EA

Anti-mouse IgG (goat) HRP

100 µLNEF822E001EA

Anti-human IgG (goat)* HRP

1 mL

NEF802001EA

Anti-DNP-HRP

150 µLFP1129Antifluorescein-HRP

2x250µL

NEF710001EAStreptavidin-HRP

2x250µL

NEL750001EA

Anti-digoxigenin HRP

500 µLNEF832001EA

Anti-rabbit IgG (goat) biotin

1 mL

NEF813001EA

Anti-mouse IgG (goat) biotin

1 mL

NEF823001EA

Anti-human IgG (goat) biotin

1 mL

NEF803001EA

Anti-digoxigenin biotin

500 µLNEF833001EAStreptavidin Fluorescein

1 mL

NEL720001EAStreptavidin Texas Red®

1 mL

NEL721001EAStreptavidin Coumarin

1 mL

NEL722001EAStreptavidin-HRP

2x250µL

NEL750001EAStreptavidin-AP

2x250µL

NEL751001EATSA Blocking Reagent
Horseradish Peroxidase Reagents

Biotin Conjugates

Labeled Streptavidin

Chromogens
BCIP/NBT SubstrateDAB Substrate

For detection of Alkaline
Phosphatase
For detection of Horseradish
Peroxidase

NEL937001PK
NEL938001EA

Alkaline Phosphatase Reagents
Anti-Mouse IgG (Goat), AP-Labeled

1 mL

NEF814001EA

Anti-Rabbit IgG (Goat), AP-Labeled

1 mL

NEF824001EAStreptavidin- AP Conjugate

2x250µL

NEL751001EA

Antifluorescein-AP Conjugate

2x250µL

NEF709001PK

Anti-DNP-AP

150 µLFP113111

Hapten Labeled Deoxynucleotides (25 nmol, for labeling of ISH probes)
3-Amino-3-Deoxydigoxigenin-9-dCTP

25 nmol

NEL562001EA

Biotin-11-dATP

25 nmol

NEL540001EA

Biotin-11-dCTP

25 nmol

NEL538001EA

Biotin-11-dGTP

25 nmol

NEL541001EA

Biotin-11-dUTP

25 nmol

NEL539001EA

DNP-11-dUTP

25 nmol

NEL551001EA

Fluorescein-12-dATP

25 nmol

NEL465001EA

Fluorescein-12-dCTP

25 nmol

NEL424001EA

Fluorescein-12-dGTP

25 nmol

NEL429001EA

Fluorescein-12-dUTP

25 nmol

NEL413001EA

Hapten Labeled Ribonucleotides (25 nmol, for labeling of ISH probes)
Biotin-11-ATP

250 nmol

NEL544001EA

Biotin-11-CTP

250 nmol

NEL542001EA

Biotin-11-GTP

250 nmol

NEL545001EA

Biotin-11-UTP

250 nmol

NEL543001EA

Fluorescein-12-ATP

250 nmol

NEL439001EA

Fluorescein-12-CTP

250 nmol

NEL434001EA

Fluorescein-12-GTP

250 nmol

NEL496001EA

Fluorescein-12-UTP

250 nmol

NEL414001EATSA and its use are protected under U.S. Patents 5,688,966, 5,863,748, 5,767,287, 6,372,937, 6,399,299, 6,593,100, 6,617,125 and patents pending, and foreign
equivalents thereof..
For Research use only. This product is distributed and sold for research purposes only by the end-user in the research market, and, to that extent, by purchasing this productthe end-user is granted a limited license to use this product for research use only. This product is not intended for diagnostic or therapeutic use and no license or right isgranted for use of this product for diagnostic or therapeutic purposes. Purchase does not include or carry any right or license to use, develop or otherwise exploit this productcommercially. Any commercial use, development or exploitation of this product without the express prior written authorization of PerkinElmer is strictly prohibited and may
constitute infringement of the intellectual property rights of PerkinElmer under the aforementioned patents.
TSA is a registered trademark of PerkinElmer. Other trademarks are property of their respective owners.

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